Throughout plant evolution the egg cell has become progressively less exposed to fertilization by the male gametophyte. Indeed, the defining feature of angiosperms is the enclosure of the ovule within a carpel. This presents a problem of accessibility for pollen tubes (Crawford, B. C. and Yanofsky, M. F., Curr Biol 18:R972-8 (2008); Williams, J. H., American Journal of Botany 96:144 (2009)). Not only are ovules distant from the point of pollen germination, in some angiosperms, such as Arabidopsis, the ovule is buried within the layers of tissue of the carpel. Additionally, the pollination and fertilization process occurs more rapidly in angiosperms compared with gymnosperms (Williams, J. H., Proc Natl Acad Sci USA 105:11259-63 (2008); Williams, J. H., American Journal of Botany 96:144 (2009)). To overcome these difficulties, angiosperms have developed a unique set of tissues to assist pollen tubes in reaching the ovules. These tissues are collectively known as the reproductive tract and consist of the stigma, style, transmitting tract and funiculus (FIG. 1A,B).
Pollen grains initially contact and germinate on elongated papillary cells of the stigma. Once germinated, they develop into pollen tubes that grow into the style, the connecting tissue between the stigma and ovary chamber (FIG. 1A,B). In Arabidopsis thaliana, pollen tubes must grow through the short, enclosed style to reach the ovary (Lennon, K. et al., Sexual Plant Reproduction 11:49-59 (1998)). In other angiosperms, however, such as lily, pollen tubes can grow on the surface of an open style to reach the ovary (Kim, S. et al., Proc Natl Acad Sci USA 100:16125-30 (2003)). The closed style of Arabidopsis contains the start of the transmitting tract, a pathway for pollen tube growth that undergoes programmed cell death (Crawford, B. C. et al., Curr Biol 17:1101-8 (2007)). Transmitting tract tissue develops in the center of the septum between the two fused carpels (FIG. 1A) and connects the style to bottom of the ovary chamber (FIG. 1B). Pollen tubes grow basally through the transmitting tract and exit laterally onto the septum epidermis, whereupon they grow towards and upon funiculi to reach ovules. The funiculus develops at the boundary of the septum and carpel walls and connects the ovules to the carpel (FIG. 1A). At the end of the funiculus, pollen tubes enter the micropyle to fertilize the egg cell.
The reproductive tract is essential for successful fertilization of ovules by pollen, and seed set is reduced in mutants that interfere with reproductive tract development (Alvarez, J. and Smyth, D. R., Int. J. Plant Sci. 163:17-41 (2002); Gremski, K. et al., Development 134:3593-601 (2007); Heisler, M. G. et al., Development 128:1089-98 (2001)). Pollen tubes also target ovules more efficiently in vitro if they have first grown through stigma and style (Palanivelu, R. and Preuss, D. BMC Plant Biol 6:7 (2006)).
The HECATE (HEC1, HEC2, HEC3) and SPATULA (SPT) genes encode putative bHLH transcription factors that play key roles in reproductive tract development since they control overall growth of the stigma, style and transmitting tract. Mutations in these genes lead to varying degrees of reduced fertility, and while HEC2-RNAi hec1 hec3 mutants are completely infertile, spt mutants show moderate infertility (Alvarez, J. and Smyth, D. R., Int. J. Plant Sci. 163:17-41 (2002); Gremski, K. et al., Development 134:3593-601 (2007); Heisler, M. G. et al., Development 128:1089-98 (2001)). HEC and SPT proteins have been shown to interact, suggesting that they act together to control development. Although the entire reproductive tract is affected in these mutants, mutations in other genes show more specific defects in reproductive tract differentiation. The NO TRANSMITTING TRACT (NTT) gene, for example, is required for normal differentiation of the ovary transmitting tract. Transmitting tract cells normally produce an extracellular matrix (ECM) containing a mixture of glycoproteins, glycolipids and polysaccharides Lennon, K. et al., Sexual Plant Reproduction 11:49-59 (1998)). The specific contribution of ECM to pollen tube growth is unknown, but growth studies have demonstrated that pollen tubes grow faster in vivo than in vitro, and this has been speculated to be due to interactions with ECM (Palanivelu, R. and Preuss, D. BMC Plant Biol 6:7 (2006)). Transmitting tract cells also undergo a process of programmed cell death essential for efficient pollen tube growth. In the ntt mutant, both ECM production and cell death are absent from the ovary transmitting tract. As a result, pollen tubes progress normally through the style but have great difficulty entering the ovary and the basal ovules remain unfertilized (Crawford, B. C. et al., Curr Biol 17:1101-8 (2007)).